Β-barrel Proteins Research Articles

Characterization of intermediate states in the fibrillogenesis of odorant-binding proteins

The transition of β-barrel proteins from a soluble to an amyloid form is biologically significant in some cases but may lead to functional activity loss. In particular, odorant-binding proteins' (OBPs) fibrils are unable to bind odorant molecules potentially contributing to olfactory dysfunction. As shown previously, OBPs' fibrillogenesis is initiated by uncoupling of protein C-terminal fragment from the β-barrel and exposing amyloidogenic sites. However, further structural transformations of OBPs are not fully understood. Here we first identified two intermediate aggregated states of OBPs: one formed by intact β-barrels, nontoxic to mammalian cells and easily dissociated by boiling and detergents; the other is prefibrillar, formed by degraded β-barrels with restructured β-strands, having almost comparable cytotoxicity but lower stability compared to mature amyloids. Obtained results revealed the β-barrel “opening” and alignment in register of its β-strands early in aggregation. Comparing the aggregation processes of bovine OBP and its mutant variant differing in the C-terminal fragment mobility showed the influence of its dynamics/orientation on the conjugation of amyloidogenic regions triggering fibril formation. The characterized properties and formation mechanisms of intermediate states in amyloidogenesis of β-barrel proteins are relevant for finding ways to prevent pathological aggregation and identifying ways to regulate the physiological fibrils assembly/disassembly.

LD-transpeptidation is crucial for fitness and polar growth in Agrobacterium tumefaciens.

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

Native β-barrel substrates pass through two shared intermediates during folding on the BAM complex.

The assembly of β-barrel proteins into membranes is mediated by the evolutionarily conserved β-barrel assembly machine (BAM) complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of β-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate β-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between β-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges.

The discovery and structural basis of two distinct state-dependent inhibitors of BamA

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins.

Biogenesis of mitochondrial β-barrel membrane proteins.

β-barrel membrane proteins in the mitochondrial outer membrane are crucial for mediating the metabolite exchange between the cytosol and the mitochondrial intermembrane space. In addition, the β-barrel membrane protein subunit Tom40 of the translocase of the outer membrane (TOM) is essential for the import of the vast majority of mitochondrial proteins encoded in the nucleus. The sorting and assembly machinery (SAM) in the outer membrane is required for the membrane insertion of mitochondrial β-barrel proteins. The core subunit Sam50, which has been conserved from bacteria to humans, is itself a β-barrel protein. The β-strands of β-barrel precursor proteins are assembled at the Sam50 lateral gate forming a Sam50-preprotein hybrid barrel. The assembled precursor β-barrel is finally released into the outer mitochondrial membrane by displacement of the nascent β-barrel, termed the β-barrel switching mechanism. SAM forms supercomplexes with TOM and forms a mitochondrial outer-to-inner membrane contact site with the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. SAM shares subunits with the ER-mitochondria encounter structure (ERMES), which forms a membrane contact site between the mitochondrial outer membrane and the endoplasmic reticulum. Therefore, β-barrel membrane protein biogenesis is closely connected to general mitochondrial protein and lipid biogenesis and plays a central role in mitochondrial maintenance.

Insights into VDAC Gating: Room-Temperature X-ray Crystal Structure of mVDAC-1.

The voltage-dependent anion channel (VDAC) is a crucial mitochondrial protein that facilitates ion and metabolite exchange between mitochondria and the cytosol. Initially characterized over three decades ago, the structure of VDAC-1 was resolved in 2008, revealing a novel β-barrel protein architecture. This study presents the first room-temperature crystal structure of mouse VDAC-1 (mVDAC-1), which is a significant step toward understanding the channel's gating mechanism. The new structure, obtained at a 3.3 Å resolution, demonstrates notable differences from the previously determined cryogenic structure, particularly in the loop regions, which may be critical for the transition between the 'open' and 'closed' states of VDAC-1. Comparative analysis of the root-mean-square deviation (R.M.S.D.) and B-factors between the cryogenic and room-temperature structures suggests that these conformational differences, although subtle, are important for VDAC's functional transitions. The application of electric field-stimulated X-ray crystallography (EF-X) is proposed as a future direction to resolve the 'closed' state of VDAC-1 by inducing voltage-driven conformational changes in order to elucidate the dynamic gating mechanism of VDAC-1. Our findings have profound implications for understanding the molecular basis of VDAC's role in mitochondrial function and its regulation under physiological conditions.

β-barrel proteins dictate the effect of core oligosaccharide composition on outer membrane mechanics.

The outer membrane is the defining cellular structure of Gram-negative bacteria, a group that contains many important pathogens like Escherichia coli , Vibrio cholerae , and Pseudomonas aeruginosa . One role of the outer membrane is to block the uptake of small molecules like antibiotics. However, it is becoming increasingly clear that it also functions as a structural exoskeleton that is critical for the cell's ability to cope with internal and external mechanical forces. Here, we carefully dissect the molecular basis for the load-bearing capacity of the outer membrane by screening a set of mutants with a new cell biophysics assay.

The Assembly of the Inverse Autotransporter Protein YeeJ is Driven by its C-terminal β-strand

Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a β-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the β1- and the β12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the β1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the β1-strand with the β3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the β12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the β-signals of classical autotransporter proteins and outer membrane β-barrel proteins in general. We therefore propose that the β12-strand of YeeJ acts as its β-signal and that the assembly of the YeeJ β-barrel is driven by its C-terminal β-strand, like in most other autotransporter proteins, despite its inverted domain organization.

Pore-Forming VDAC Proteins of the Outer Mitochondrial Membrane: Regulation and Pathophysiological Role.

Voltage-dependent anion channels (VDAC1-3) of the outer mitochondrial membrane are a family of pore-forming β-barrel proteins that carry out controlled "filtration" of small molecules and ions between the cytoplasm and mitochondria. Due to the conformational transitions between the closed and open states and interaction with cytoplasmic and mitochondrial proteins, VDACs not only regulate the mitochondrial membrane permeability for major metabolites and ions, but also participate in the control of essential intracellular processes and pathological conditions. This review discusses novel data on the molecular structure, regulatory mechanisms, and pathophysiological role of VDAC proteins, as well as future directions in this area of research.

Grubbs-Hoveyda catalysts conjugated to a β-barrel protein: Effect of halide substitution on aqueous olefin metathesis activity

The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered β-barrel protein nitrobindin (NB4exp) on metathesis activity in aqueous media was studied. Maleimide tagged dibromido and diiodido derivates of the GHII catalyst were synthesized and covalently conjugated to NB4exp. The biohybrid catalysts were characterized spectroscopically confirming the structural integrity. When the two chloride substituents at ruthenium center were exchanged against bromide and iodide, the diiodo derivative was found to show significantly higher catalytic activity in ring-closing metathesis of α,ω-diolefins, whereas the dibromido derivative was less efficient when compared with the parent dichlorido catalyst. Using the diiodido catalyst, high turnover numbers of up to 75 were observed for ring-closing metathesis (RCM) yielding unsaturated six- and seven-membered N-heterocycles.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The sorting and assembly machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing systemcomplex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopyand computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa.

Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

CRUMPLED LEAF supports plastid OUTER ENVELOPE PROTEIN OF 80 KDA complex formation in Arabidopsis.

Embedded β-barrel proteins in the outer envelope membrane mediate most cellular trafficking between the cytoplasm and plastids. Although the TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 75-V (TOC75-V)/OUTER ENVELOPE PROTEIN OF 80 KDA (OEP80) complex has been implicated in the insertion and assembly of β-barrel proteins in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts, relatively little is known about this process. CRUMPLED LEAF (CRL) encodes a chloroplast outer envelope membrane-localized protein, and its loss-of-function mutation results in pleiotropic defects, including altered plant morphogenesis, growth retardation, suppression of plastid division, and spontaneous light intensity-dependent localized cell death. A suppressor screen conducted on mutagenized crl mutants revealed that a missense mutation in OEP80 suppresses the pleiotropic defects of crl. Furthermore, we found that OEP80 complex formation is compromised in crl. Additionally, we demonstrated that CRL interacts with OEP80 in vivo and that a portion of CRL is present at the same molecular weight as the OEP80 complex. Our results suggest that CRL interacts with OEP80 to facilitate its complex formation. CRL is involved in plastid protein import; therefore, the pleiotropic defects in crl are likely due to the combined effects of decreased plastid protein import and altered membrane integration of β-barrel proteins in the outer envelope membrane. This study sheds light on the mechanisms that allow β-barrel protein integration into the plastid outer envelope membrane and the importance of this finding for plant cellular processes.

Recent Advances in Modeling Membrane β-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand β-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5years, in modeling β-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of β-barrel protein modeling: how different types of molecules can diffuse through β-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how β-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Modular Assembly of Mitochondrial β-Barrel Proteins.

Mitochondrial β-barrel proteins fulfill crucial roles in the biogenesis and function of the cell organelle. They mediate the import and membrane insertion of proteins and transport of small metabolites and ions. All β-barrel proteins are made as precursors on cytosolic ribosomes and are imported into mitochondria. The β-barrel proteins fold and assemble with partner proteins in the outer membrane. The in vitro import of radiolabelled proteins into isolated mitochondria is a powerful tool to investigate the import of β-barrel proteins, the folding of the β-barrel proteins, and their assembly into protein complexes. Altogether, the in vitro import assay is a versatile and crucial assay to analyze the mechanisms of the biogenesis of mitochondrial β-barrel proteins.

Recombinant Expression and Overproduction of Transmembrane β-Barrel Proteins.

Transmembrane β-barrel proteins reside in the outer membrane of Gram-negative bacteria and are thus in direct contact with the environment. Because of that, they are involved in many key processes stretching from cellular survival to virulence. Hence, they are an attractive target for the development of novel antimicrobials, in addition to being of fundamental biological interest. To study this class of proteins, they are often required to be expressed in Escherichia coli. Recombinant expression of β-barrel proteins can be achieved using two fundamentally different strategies. The first alternative uses a complete coding sequence that includes a signal peptide for targeting the protein to its native cellular location, the bacterial outer membrane. The second alternative omits the signal peptide in the gene, leading to mislocalization and aggregation of the protein in the bacterial cytoplasm. These aggregates, called inclusion bodies, can be solubilized and the protein can be folded into its native form in vitro. In this chapter, we present example protocols for both strategies and discuss their advantages and disadvantages.

Analysis of Transmembrane β-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded β-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. β-barrels are highly structured domains formed by a series of antiparallel β-strands. Each β-strand is locked in position by hydrogen bonds between its polypeptide backbone andthose of thetwo neighbouring strands in the barrel structure. Some transmembraneβ-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembraneβ-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as arapid method to investigate the folding of transmembrane β-barrels.

Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial β-Barrel Assembly Machine.

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a β-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different β-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling β-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.

The Name Is Barrel, β-Barrel.

β-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel β-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are β-barrel outer membrane proteins (OMPs). β-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of β-barrel OMPs, how they are folded into the membrane, and the future of β-barrel OMP research and its applications.

Tracking the Activity and Position of Mitochondrial β-Barrel Proteins.

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.